Compositions for treating biofilms and methods for using same

ABSTRACT

Compositions containing a surface active agent and a sub-lethal amount of an antimicrobial agent and methods for using such compositions are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/524,332 entitled “Compositions for Treating Biofilms and Methods forUsing Same,” filed Oct. 27, 2014, which is a divisional of U.S. patentapplication Ser. No. 13/685,444 entitled “Compositions for TreatingBiofilms and Methods for Using Same,” filed Nov. 26, 2012 which is acontinuation of U.S. patent application Ser. No. 12/033,692 entitled“Compositions. For Treating Biofilms And Methods For Using Same,” filedFeb. 19, 2008 which claims priority to and benefit of U.S. ProvisionalApplication No. 60/890,535 entitled “Treating Biofilm with SurfactantCompositions” filed on Feb. 19, 2007 the entire contents of which arehereby incorporated by reference in their entireties.

GOVERNMENT INTERESTS

Not applicable

PARTIES TO A JOINT RESEARCH AGREEMENT

Not applicable

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not applicable

BACKGROUND

1. Field of Invention

Not Applicable

2. Description of Related Art

Bacteria and fungi form biofilms under certain conditions. When a groupof bacteria or fungi accumulate on a surface and reach a particular celldensity, they begin to secrete a polymeric substance that consists ofpolysaccharides, proteins and DNA and form a matrix in which thebacterial or fungal cells are entrenched. The multi-cellular aggregatesor biofilms allow for individual bacterial or fungal cells or coloniesof bacterial or fungal cells to exhibit coordinated behavior and conferupon the microorganism advantages including, for example, resistance toantibiotics and host immune systems. More specifically, biofilms arestructured to allow respiration and fluid and nutrient exchange whilepreventing access of host immune cells such as phagocytes and preventinginhibitory or lytic concentrations of antimicrobials from reaching themicroorganisms. As a result of these properties, infections that resultfrom biofilm formation are notoriously difficult to eradicate andrequire the use of high concentrations of antimicrobial agents, removalof tissue, debridement of affected tissues and combination of thesetreatments.

Biofilm formation appears to be governed by secretion and detection ofautoinducer molecules in a process referred to as “quorum sensing.” Bythis process, as quorum sensing autoinducer molecules begin toaccumulate in the area surrounding the microorganisms, themicroorganisms begin to undergo a number of physiological changes whichallow for formation of the extracellular biofilm. For example, as aresult of quorum sensing autoinducer accumulation, the microorganismsmay begin surface attachment, extracellular polymer production,biosurfactant production, sporulation, bioluminescence and secretion ofnutrient sequesteration molecules and virulence factors among othereffects resulting in biofilm formation.

Because of the properties provided by the polymeric matrix and thephysiological changes exhibited by microorganisms in a biofilm,microorganisms in a biofilm are typically less susceptible toantibiotics, antimicrobials and biocides. In some cases, bacteria in abiofilm can be up to 4,000 times more resistant (i.e., less susceptible)than the same organism in a free-floating (planktonic) state.Comparisons of minimum inhibitory concentration (MIC) which describesthe amount of an active agent delivered to planktonic microorganismsnecessary to inhibit biofilm formation and minimum biofilm eradicationconcentration (MBEC) which describes the minimum concentration of anactive agent delivered to a biofilm necessary to inhibit or eradicatebiofilm growth illustrate the differences in susceptibility from theplanktonic bacteria to those in a biofilm and show that biofilm formingbacteria are much less susceptible to antimicrobial agents at standardtherapeutic concentrations. Moreover, using an MIC concentration ofantibiotic in a biofilm infection can inadvertently expose the biofilmto a sub-lethal dose of antibiotics which may increase the likelihood ofthe exposed biofilm forming microorganisms developing resistance to theantimicrobial agent which can have grave consequences for effectivetreatment of the biofilm.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention described herein are directed to a methodfor treating a microbial biofilm on a patient including the steps ofcontacting the microbial biofilm with a composition comprising a surfaceactive agent and a sub-lethal amount of an antimicrobial agent.

In some embodiments, the antimicrobial agent may be an antibacterial,antifungal, antiviral or combinations thereof, and in others, theantimicrobial agent may be furaltadone, furazolium chloride, nifuradene,nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol,nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran,nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin,silver sulfadiazine, nanocrystalline silver, ionic silver, honey,iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidineor combinations thereof. In certain embodiments, a sub-lethal amount ofan antimicrobial agent may be a standard therapeutically effectiveamount. In particular embodiments, the antimicrobial agent may be silversulfadiazine and the sub-lethal amount may be less than about 1% byweight of the composition, less than about 0.95% by weight of thecomposition, or less than about 0.75% by weight of the composition.

The surface active agent of embodiments may be a poloxamer, meroxapol,poloxamine or combinations thereof. In some embodiments, the surfaceactive agent may be a poloxamer 127, poloxamer 188, poloxamer 237,poloxamer 335, poloxamer 407 or combinations thereof, and in certainembodiments, the surface active agent may be poloxamer 188.

The step of contacting the microbial biofilm in various embodiments mayinclude applying the composition to a wound. In some embodiments, thestep of contacting the microbial biofilm may include administering thecomposition topically, and in particular embodiments, administering thecomposition topically may be selected from administering by hand,administering by an extruder, spray delivery, applying a dressingincluding the composition or combinations thereof. In other embodiments,the step of contacting the microbial biofilm may include contactingtissue from the patient that is outside the patient, and in still otherembodiments, the step of contacting may include applying the compositionto a dressing prior to applying the dressing to the patient.

In some embodiments, the patient may be afflicted with an injury, and incertain embodiments, the injury may be a burn, abrasion, cut, scrape,denuding tissue injury or combinations thereof. In other embodiments,the patient may be afflicted with a chronic wound, and in particularembodiments, the chronic wound may be a venous ulcer, diabetic ulcer,arterial ulcer, pressure ulcer, radiation ulcer, traumatic wound,non-healing wound or combinations thereof.

Embodiments of the invention also include a method for preventing amicrobial biofilm on a patient including the steps of administering acomposition comprising a surface active agent and a sub-lethal amount ofan antimicrobial agent to an injury.

In some embodiments, the antimicrobial agent may be an antibacterial,antifungal, antiviral or combinations thereof, and in others, theantimicrobial agent may be furaltadone, furazolium chloride, nifuradene,nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol,nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran,nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin,silver sulfadiazine, nanocrystalline silver, ionic silver, honey,iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidineor combinations thereof. In particular embodiments, the antimicrobialagent may be silver sulfadiazine and the sub-lethal amount may be lessthan about 1% by weight of the composition, less than about 0.95% byweight of the composition, or less than about 0.75% by weight of thecomposition.

The surface active agent of embodiments may be a poloxamer, meroxapol,poloxamine or combinations thereof. In some embodiments, the surfaceactive agent may be a poloxamer 127, poloxamer 188, poloxamer 237,poloxamer 335, poloxamer 407 or combinations thereof, and in certainembodiments, the surface active agent may be poloxamer 188.

The step of contacting the microbial biofilm in various embodiments mayinclude applying the composition to a wound. In some embodiments, thestep of contacting the microbial biofilm may include administering thecomposition topically, and in particular embodiments, administering thecomposition topically may be selected from administering by hand,administering by an extruder, spray delivery, applying a dressingincluding the composition or combinations thereof. In other embodiments,the step of contacting the microbial biofilm may include contactingtissue from the patient that is outside the patient, and in still otherembodiments, the step of contacting may include applying the compositionto a dressing prior to applying the dressing to the patient.

In particular embodiments, the composition may be administered whilebiofilm forming pathogens are in a planktonic state.

Further embodiments of the invention include a method for treating orpreventing a microbial biofilm in a patient including the steps ofadministering a first composition comprising a surface active agent toan injury wherein the first composition does not contain anantimicrobial agent and administering a second composition comprising asurface active agent and a sub-lethal amount of an antimicrobial agentto the injury. In certain embodiments, the first composition may beadministered before the second composition, and in some embodiments, thesecond composition may be applied to a dressing prior to itsadministration.

In various embodiments, the antimicrobial agent of the secondcomposition may be an antibacterial, an antifungal, an antiviral or acombination thereof, and in some embodiments, the antimicrobial agent ofthe second composition may be furaltadone, furazolium chloride,nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine,nifurtoinol, nitrofurantoin, furazolidone,2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide,nitrofurazone, nystatin, polymyxin, silver sulfadiazine silver salts,nanocrystalline silver, ionic silver, iodine, benzalkonium chloride,alcohol, hydrogen peroxide, chlorhexidine or combinations thereof. Inparticular embodiments, the antimicrobial agent may be silversulfadiazine and the sub-lethal amount may be less than about 1% byweight of the composition, less than about 0.95% by weight of thecomposition, or less than about 0.75% by weight of the composition.

The surface active agents in the first and second composition of variousembodiments may be surfactants. In some embodiments, the surface activeagents in the first and second compositions may independently bepoloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer407 or combinations thereof. In particular embodiments, the surfaceactive agents in the first and second compositions may be the same, andin at least one embodiment, surface active agents in the first andsecond compositions may be poloxamer 188.

Embodiments also include a composition for treating or preventing amicrobial biofilm including a surface active agent and a sub-lethalamount of an antimicrobial agent.

In some embodiments, the antimicrobial agent may be an antibacterial,antifungal, antiviral or combinations thereof, and in others, theantimicrobial agent may be furaltadone, furazolium chloride, nifuradene,nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol,nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran,nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin,silver sulfadiazine, nanocrystalline silver, ionic silver, honey,iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidineor combinations thereof. In certain embodiments, a sub-lethal amount ofan antimicrobial agent may a standard therapeutically effective amount.In particular embodiments, the antimicrobial agent may be silversulfadiazine and the sub-lethal amount may be less than about 1% byweight of the composition, less than about 0.95% by weight of thecomposition, or less than about 0.75% by weight of the composition.

The surface active agent of various embodiments may be a poloxamer,meroxapol, poloxamine or combinations thereof. In embodiments in whichthe surface active agent is a poloxamer, the poloxamer may be apoloxamer 101, poloxamer 105, poloxamer 105 benzoate, poloxamer 108,poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer182, poloxamer 182 dibenzoate, poloxamer 183, poloxamer 184, poloxamer185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217,poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331,poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer401, poloxamer 402, poloxamer 403 and poloxamer 407 or combinationsthereof. Agent is a copolymer selected from poloxamer 127, poloxamer188, poloxamer 237, poloxamer 335, poloxamer 407 or combinationsthereof. In particular embodiments, the surface active agent may bepoloxamer 188. In embodiments in which the surface active agent is ameroxapol, the meroxapol may be a meroxapol 105, meroxapol 108,meroxapol 171, meroxapol 172, meroxapol 174, meroxapol 178, meroxapol251, meroxapol 252, meroxapol 254, meroxapol 258, meroxapol 311,meroxapol 312, meroxapol 314 or combinations thereof. In embodiments inwhich the surface active agent is a poloxamine, the poloxamine may be apoloxamine 304, poloxamine 504, poloxamine 701, poloxamine 702,poloxamine 704, poloxamine 707, poloxamine 901, poloxamine 904,poloxamine 908, poloxamine 1101, poloxamine 1102, poloxamine 1104,poloxamine 1301, poloxamine 1302, poloxamine 1304, poloxamine 1307,poloxamine 1501, poloxamine 1502, poloxamine 1504, poloxamine 1508 orcombinations thereof.

In some embodiments, the composition may include one or more secondaryactive agents, and in others, the composition may include one or moreadditives. In particular embodiments, the composition may furtherinclude a solvent.

Embodiments of the invention also include a dressing for treating orpreventing a microbial biofilm including a first composition layercomprising a surface active agent wherein the first gel layer does notinclude an antimicrobial agent, a second composition layer comprising asurface active agent and a sub-lethal amount of an antimicrobial agent,and a dressing material supporting said first and second compositionlayers. In some such embodiments, the second composition layer may belocated between the first composition layer and the dressing material.

In some embodiments, the antimicrobial agent may be an antibacterial,antifungal, antiviral or combinations thereof, and in others, theantimicrobial agent may be furaltadone, furazolium chloride, nifuradene,nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol,nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran,nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin,silver sulfadiazine, nanocrystalline silver, ionic silver, honey,iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidineor combinations thereof. In particular embodiments, the antimicrobialagent may be silver sulfadiazine and the sub-lethal amount may be lessthan about 1% by weight of the composition, less than about 0.95% byweight of the composition, or less than about 0.75% by weight of thecomposition.

The surface active agent of embodiments may be a poloxamer, meroxapol,poloxamine or combinations thereof. In some embodiments, the surfaceactive agent may be a poloxamer 127, poloxamer 188, poloxamer 237,poloxamer 335, poloxamer 407 or combinations thereof, and in certainembodiments, the surface active agent may be poloxamer 188.

In some embodiments, the surface active agent in the first and secondcompositions may be the same. In other embodiments, the dressing mayfurther include a spacer material layer between said first and secondcomposition layers. The spacer material of such embodiments may fully orpartially lose integrity upon application of the dressing to a patient.In particular embodiments, the second composition layer may impregnatethe dressing material.

Other embodiments of the invention also include a method for preventinga microbial biofilm on a patient including the steps of administering acomposition including a surface active agent and a sub-lethal amount ofan antimicrobial agent to a wound prior to infection. Still otherembodiments of the invention include a method for preventing a microbialbiofilm on a patient including the steps of administering a compositionincluding a surface active agent and a sub-lethal amount of anantimicrobial agent to a wound while biofilm forming bacteria are in aplanktonic state. Yet other embodiments of the invention include amethod for preventing a microbial biofilm on a patient including thesteps of administering a composition comprising a surface active agentand a sub-lethal amount of an antimicrobial agent to a wound prior toinfection wherein the composition is administered within 10 hours of theinjury. Further embodiments of the invention include a method fortreating or preventing a microbial biofilm on a patient including thesteps of administering a composition comprising a surface active agentto a wound.

DESCRIPTION OF DRAWINGS

Not Applicable

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

It must be noted that, as used herein, and in the appended claims, thesingular forms “a”, “an” and “the” include plural reference unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the present invention, thepreferred methods are now described. All publications and referencesmentioned herein are incorporated by reference. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Optional” or “optionally” may be taken to mean that the subsequentlydescribed structure, event or circumstance may or may not occur, andthat the description includes instances where the event occurs andinstances where it does not.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. “Administering” acomposition may be accomplished by injection, infusion, or by eithermethod in combination with other known techniques. Such combinationtechniques include heating, radiation and ultrasound.

The terms “treat,” “treating” or treatment” generally mean the exposureof a living organism to one or more physical, chemical or psychologicalentities or stimuli that may prevent, improve or ameliorate a diseasedstate. These terms are also meant to encompass exposing an inanimateobject (e.g., biomaterial) to physical or chemical entities or stimulithat may enhance the object's capacity to alter a disease process inliving organisms which is contacted by the object. For example, treatinga biomaterial may include applying a composition to a surface of abiomaterial to enhance resistance to biofilm development, dissolve anexisting biofilm and/or deliver a therapeutic to the biomaterials.Biomaterial treatment may encompass treating an entry site for cathetersin the body of a patient which allow access of the catheter to, forexample, blood vessels, body cavities, cerebrospinal space and the like.

The term “indication”, as used herein, refers to a medical condition orsymptoms associated with a medical condition, such as biofilm infection.For example, redness and swelling of tissue surrounding an injury may bean indication of subject in a diseased state.

The term “target”, as used herein, refers to the material for whichdeactivation, rupture, disruption or destruction is desired. Forexample, infectious microorganisms or biofilms may be consideredundesirable material in an injured subject and may be a target fortherapy.

Generally speaking, the term “tissue” refers to any aggregation ofsimilarly specialized cells which are united in the performance of aparticular function. For example, skin may be considered a tissue.

The term “diseased tissue”, as used herein, refers to tissue or cellsassociated with an injury that has become infected with microorganisms,and in particular, microorganisms capable of forming a biofilm or tissueon which a biofilm has formed.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the tissue to which it is being provided, applied oradministered. For example, eradication of the bio film would improve theindications of the injury.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient.

A “therapeutically effective amount” or “effective amount” of acomposition as used herein is a predetermined amount calculated toachieve the desired effect. For example, a “therapeutically effectiveamount” of a composition of the invention may achieve one or more ofpreventing formation of a biofilm, disrupting preformed biofilm and/orenabling contact of one or more therapeutic agents with themicroorganism responsible for the biofilm or enabling augmentation ofthe state of the tissue underlying the biofilm so as to ameliorate thedisease state.

A “biofilm” as used herein describes an aggregate of microorganisms thatexhibit cooperative behavior such as, for example, secretion of apolymeric matrix that protects the microorganisms from attack by thehost immune system.

By “biomaterial” is meant, a non-drug material that can be used totreat, enhance, or replace any tissue, organ, or function in anorganism.

A “surface active agent” or “surfactant,” as used herein, may refer to asubstance that is capable of reducing the surface tension of a material.

Embodiments of the invention presented herein are generally directed tocompositions for use in preventing the formation of biofilms andtreating biofilms that have formed, methods for using such compositionsand materials including such compositions such as, for example, wounddressings, surgical equipment and syringes. Embodiments of the inventionare directed to treating biofilms both living and non-living objectsusing the compositions of the invention. Some embodiments are directedto compositions and methods useful for the treatment of a patient,living tissue or biomaterial. Compositions used in such embodiments maybe referred to as “pharmaceutical compositions” which generally refersto a composition that is meant for application on or in a patient,living tissue or a biomaterial. Similar compositions, even those havingthe same makeup, may be useful in methods for treating biofilms onobjects that are not living or utilized in the treatment of a livingbeing which are also encompassed by the invention described herein.

In various embodiments, the composition of the invention may at leastinclude a surface active agent and a sub-lethal amount or dose of anantimicrobial agent. Surface active agents are well known in the art,and suitable surface active agents for preparing the compositions of theinvention are not particularly limiting. For example, anionic, cationicor non-ionic surface active agents may be used individually or incombination. In some embodiments, non-ionic surface active agents basedon a polyol and including alkylene oxide units such as ethylene oxideand propylene oxide may be used. Such non-ionic surface active agentsinclude, but are not limited to, glycerol stearate/polyethylene glycolstearate co-polymers marketed under the trade name, ARLACEL′ andsorbitan stearate/sugar cocoate copolymers marketed under the trade nameARLATONE′. In other embodiments, the surface active agent may be acopolymer, such as, a poloxamer, meroxapol, and poloxamine.

Poloxamers are well known in the art and generally refer to a class ofnon-ionic di-block or tri-block copolymers having a central hydrophobicchain of polyoxypropylene flanked by hydrophilic chains ofpolyoxyethylene. An exemplary tri-block poloxamer may be of generalformula:

H[OCH₂CH₂]_(a)[OCH(CH₃)CH₂]_(b)[OCH₂CH₂]_(c)OH

where a, b and c are an integer and reflect the number of ethylene oxideand propylene oxide monomers in each block. The length of each polymerblock may vary and may provide poloxamers with different properties, andpoloxamers are available in various grades. In general, poloxamers arenamed using three digits, the first two digits×100 give the approximatemolecular mass of the polyoxypropylene core and the last digit×10 givesthe percentage polyoxyethylene content in the poloxamer. For example,poloxamer 188 would be expected to contain a polyoxypropylene core of1800 g/mol and a polyethylene content of approximately 80%. Exemplarypoloxamers useful in embodiments of the invention may include, but arenot limited to, poloxamers 101, 105, 105 benzoate, 108, 122, 123, 124,181, 182, 182 dibenzoate, 183, 184, 185, 188, 212, 215, 217, 231, 234,235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, 403 and407.

Meroxapols are well known in the art and generally refer to a class ofnonionic di-block or tri-block copolymers having a central hydrophilicchain of polyoxyethylene flanked by hydrophobic chains ofpolyoxypropylene. An exemplary tri-block meroxapol may be of generalformula:

H[OCH(CH₃)CH₂]_(a)[OCH₂CH₂]_(b)[OCH(CH₃)CH₂]_(c)OH

where a, b and c are an integer and reflect the number of ethyleneoxide, and propylene oxide monomers in each block. The length of eachpolymer block may vary and may provide meroxapols with differentproperties. Meroxapols are available in various grades, and are namedusing the system described above for poloxamers. Examples of meroxapolsuseful in embodiments of the invention include, but are not limited to,meroxapols 105, 108, 171, 172, 174, 178, 251, 252, 254, 258, 311, 312and 314.

Poloxamines are well known in the art and generally refer to a class ofnonionic tri-block copolymers having a central ethylene diamine flankedon either side by polyoxyethylene-polyoxypropylene block copolymers.Such compounds conform to general formula:

H([OCH₂CH₂]_(a)[OCH(CH₃)CH₂]_(b))_(x)NCH₂CH₂N(OCH(CH₃)CH₂]_(c)[OCH₂CH₂]_(d))_(y)OH

where a, b, c and d are an integer and reflect the number of ethyleneoxide and propylene oxide monomers in eachpolyoxyethylene-polyoxypropylene copolymer block and x and y areintegers and reflect the number of polyoxyethylene-polyoxypropylenecopolymer blocks in each block. Poloxamines are available in differentgrades and are named using the system described above for poloxamers.Examples of poloxamines useful in embodiments of the invention include,but are not limited to poloxamines 304, 504, 701, 702, 704, 707, 901,904, 908, 1101, 1102, 1104, 1301, 1302, 1304, 1307, 1501, 1502, 1504 and1508.

The number average molecular weight (M_(n)) of surface active agents,and particularly copolymer surface active agents of embodiments may varyand may be, for example, from about 600 to about 20,000, in someembodiments from about 600 to about 10,000, and in other embodiments,from about 1,000 to about 9,000. In particular embodiments, the M_(n)may be from about 5,000 to about 8,500. The weight of hydrophobic groupsmay also vary and may be from about 45% to about 95% by weight of thecopolymer.

The surface active agent of embodiments may be hydrated by mixing asuitable dry formulation of a surface active agent with a solvent suchas, for example, water. Surface active agents may be hydrated by anymethod known in the art. For example, in some embodiments a surfaceactive agent including a copolymer (e.g., a poloxamer) can be hydratedby cooling a mixture of the copolymer and water to an appropriatetemperature such as, for example, about 0° F. to about 50° F. for atleast 4 hours. The ratio of surface active agent to solvent may varyamong embodiments and may be, for example, about 0.01% to about 99.99%w/w, about 20% to about 90% w/w, about 30% to about 80% w/w and about40% to about 70% w/w. In particular embodiments the ratio of surfaceactive agent to water may be 1:1 or about 50% w/w. Such embodiments aredescribed in greater detail in U.S. Pat. No. 5,635,540, the contents ofwhich are incorporated herein by reference in its entirety.

Embodiments of the invention include the use of a surface active agentsuch as those described above to treat or eradicate a biofilm orplanktonic biofilm forming microorganisms. In such embodiments, acomposition including a surface active agent may be applied to a surfaceor wound where biofilm exists or where planktonic biofilm formingmicroorganism may be present and there is a high likelihood of a biofilmforming. The composition may reduce or eliminate an existing biofilm orinhibit the growth of or eradicate biofilm forming microorganismsthereby reducing or eliminating the formation a biofilm. Suchcompositions may be applied to a wound on a patient or applied to asurface, such as, an operating table or surgical instruments withsimilar efficacy.

In certain embodiments, the surface active agent composition may furtherinclude one or more therapeutic agents (e.g., 2, 3, or 4 therapeuticagents may be added to a surface active agent). In general, atherapeutic agent may be added to a surface active agent after thesurface active agent has been hydrated. However, in some embodiments, adry surface active agent may be mixed with a dry therapeutic agent andthe surface active agent may be hydrated following such mixing. In otherembodiments, a dry mixture of surface active agent and therapeutic agentmay be prepared and stored for a period of time before the surfaceactive agent is hydrated. Any therapeutic agent known in the art may bemixed with the surface active agent to prepare a composition encompassedby the invention. For example, useful therapeutic agents may include,but are not limited to, antimicrobials, steroids (e.g., hydrocortisone,triamcinolone), pain medications (e.g., aspirin, an NSAID, and a localanesthetic), anti-inflammatory agents, biological cells and biologicalagents of various types and combinations thereof.

In certain embodiments, the therapeutic agent may be an antimicrobialagent. Antimicrobial agents are well known and utilized in the art andinclude antibacterial, antifungal, and antiviral agents. Examples ofantimicrobials useful in embodiments of the invention include, but arenot limited to, silver sulfadiazine, nanocrystalline silver, ionicsilver, nystatin, nystatin/triamcinolone, bacitracin, nitrofurazone,nitrofurantoin, polymyxins (e.g., colistin, surfactin, polymyxin E andpolymyxin B), doxycycline, natural and synthetic antimicrobial peptidesand combinations of agents. Antimicrobials of embodiments may alsoinclude topical antimicrobials (i.e., antiseptics), for example, silversalts, iodine, benzalkonium chloride, alcohol, hydrogen peroxide,chlorhexidine, honey and the like.

As described above, the polymer matrix formed between microorganismsforms a barrier to many antimicrobial agents. Therefore, treatment ofbiofilms and biofilm forming microorganisms generally requires one ormore doses of an antimicrobial agent that is often several times greaterthan the lethal dosage required to treat individual microorganisms thatdo not form biofilms or planktonic biofilm forming microorganisms.Moreover, it has been reported that sub-inhibitory concentrations ofantimicrobial agents may induce biofilm formation (see, for example,Frank et al. “In Vitro Effects of Antimicrobial Agents on Planktonic andBiofilm Forms of Staphylococcus lugdunensis Clinical Isolates.”Antimicrobial Agents and Chemotherapy, Mar. 2007, p. 888-895). Thus, thelethal dosage for treatment of biofilm forming microorganisms may besignificantly higher than the standard therapeutically effective amountdetermined for planktonic microorganisms (i.e., a lethal amount or alethal dosage) typically used by one of ordinary skill in the art or theamount approved by a regulatory agency (i.e., the FDA or its Europeancounterpart).

Without wishing to be bound by theory, the surface active agent used inthe context of the compositions of the invention, with or withoutadditional agents, may penetrate and/or disrupt established biofilms byweakening or dispersing the polymer matrix or portions of the polymermatrix thereby diminishing or eliminating its function in providingresilience to the entrenched microorganisms. By weakening or disruptingof the polymer matrix of the biofilm, the surface active agent mayenable the therapeutic agent to access the microorganisms of the biofilmand/or the tissues/surfaces beneath the biofilm improving delivery of atherapeutic agent to the microorganisms and/or the injured tissues.

Using this rationale, it would be expected that the amount ofantimicrobial agent necessary to treat biofilm forming microorganismswhen administered in conjunction with a surface active agent would beconsistent with the standard therapeutically effective amount. Thus, theskilled artisan would expect to use a “standard therapeuticallyeffective amount” or a “standard therapeutic dose” which as used hereinrefers to an amount of an antimicrobial agent suggested by themanufacturer or approved for clinical use by regulatory agencies suchas, for example, the Food and Drug Administration (the “FDA”) whicheffects treatment of microorganisms in planktonic form. A “standardtherapeutic amount” or “standard therapeutic dose” may also refer to anamount of an agent sufficient to reduce or eliminate planktonicmicroorganisms. As described above, biofilms are generally not effectedby antimicrobial agents provided at a standard therapeutic effectiveamount because biofilms are resistant to many antimicrobial agents.Therefore, a standard therapeutically effective amount may not representa therapeutically effective amount of an antimicrobial agent when theantimicrobial agent is used to treat a biofilm. However, the dataprovided herein surprisingly demonstrate that a standard therapeuticallyeffective amount of antimicrobial agent may be sufficient to effectivelytreat both planktonic biofilm forming microorganisms and preformedbiofilms when the microbial agent is administered in combination with asurface active agent. Moreover, these data may additionally show thatthe amount of a microbial agent required to successfully treat biofilmforming microorganism when administered in combination with a surfaceactive agent may be a “sub-lethal” amount (i.e., less than a standardtherapeutically effective amount).

As used herein the terms “sub-lethal dose” or “sub-lethal amount” referto an amount of an antimicrobial agent that is less than the standardtherapeutically effective amount. In the context of the inventiondescribed herein, a sub-lethal amount of an antimicrobial agent mayeffectively eradicate or inhibit the growth of biofilm formingmicroorganisms or pathogens, or inhibit biofilm formation or eradicateformed biofilms. For example, a sub-lethal amount of an antimicrobialagent in some embodiments may be from about 10% to greater than about50% less than the standard therapeutically effective amount or 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% or 50% less than the standardtherapeutically effective amount approved by a regulatory agency. Inother embodiments, a sub-lethal amount may be greater than 50% less thanthe standard therapeutically effective amount. In still otherembodiments, a sub-lethal may include 100% of the standardtherapeutically effective amount when the composition is used to treat abiofilm.

For example, a standard therapeutically effective amount of a silversulfadiazine is about 1.0% by weight when used in an antimicrobial creamor gel. However, when silver sulfadiazine is administered in combinationwith a surface active agent to a biofilm forming microorganism, theamount of silver sulfadiazine in the composition may be decreased belowthe standard therapeutically effective amount to, for example, less than1.0% by weight. Therefore, a sub-lethal amount of silver sulfadiazine insome embodiments, may be less than 0.95% by weight, less than 0.90% byweight, less than 0.85% by weight, less than 0.80% by weight, less than0.75% by weight, less than 0.70% by weight, less than 0.65% by weight,less than 0.60% by weight or less than 0.55% by weight of thecomposition. In other embodiments, the amount of silver sulfadiazine maybe less than 0.5% by weight of the composition. In embodiments, wheresilver sulfadiazine is used to treat a formed biofilm, a therapeuticallyeffective amount may be 100% of a therapeutically effective amount or1.0% by weight of the composition.

In another exemplary embodiment, a standard therapeutic dose ofPolymyxin B is about 10,000 units/gram in various compositions. Thus, a“sub-lethal” dose or amount of Polymyxin B may be from about 1% to about50% less than the about 10,000 units/gram standard therapeutic dose, or9,900 units/gram to 5,000 units/gram, respectively. In certainembodiments, a sub-lethal amount may be greater than 50% less than astandard therapeutic effective amount, for example, about 60% less,about 70% less or about 80% less. In embodiments where a Polymyxin B isused to treat a formed biofilm, a therapeutically effective amount maybe 100% of a therapeutically effective amount or 10,000 units/gram.

Similarly, a standard therapeutic dose of nystatin is about 4,000units/gram. Thus, a “sub-lethal” dose of nystatin may be from about 1%less (3,960 units/gram) to about 50% less (2,000 units/gram). In yetanother example, a standard therapeutically effective amount ofnitrofurantoin is about 0.3%. Therefore, a sub-lethal amount ofnitrofurantoin may be less than about 0.3%, for example, about 0.29% toabout 0.15%. In embodiments where a Polymyxin B is used to treat aformed biofilm, a therapeutically effective amount may be 100% of atherapeutically effective amount or 4,000 units/gram.

The compositions of embodiments of the invention may be administered incombination with secondary active agents, such as, for example, drugs,adjuvants, protease inhibitors or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein. In someembodiments, the secondary active agent may be administered separatelyfrom the composition, and in others, the secondary active agent may be acomponent of the compositions of the invention. For example, in certainembodiments, the composition containing a surface active agent and asub-lethal amount of an antimicrobial agent may further contain a drugfor reducing irritation or enhancing healing such as, for example, ananti-inflammatory agent, anesthetic, pain killer or steroid.

Other embodiments of compositions encompassed by the invention mayinclude additives such as stabilizers, antioxidants, osmolalityadjusting agents, buffers, pH adjusting agents, chelants, calciumchelate complexes, salts or combinations thereof. For example, in someembodiments, a stabilizer such as appropriate pharmaceutical gradesurfactants such as, TWEEN or saccharides, like dextrose, may be addedto the compositions of the invention, and in some embodiments, suchcompositions may also include conventional pharmaceutical excipientsand/or additives. For example, suitable pharmaceutical excipients mayinclude stabilizers, antioxidants, osmolality adjusting agents, buffers,and pH adjusting agents, and suitable additives may includephysiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Conventional nontoxic carriers may alsobe incorporated into such compositions and may include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate and the like. For example, about 1 to about 95% by volume or,in a further example, 25% to about 75% by volume of any of the carriersand excipients listed above may be mixed into the compositions of theinvention. Additional additives such as coloring agents, thickeners,lubricants and so on may also be added to the compositions of theinvention. Compositions of various embodiments of the invention may beprepared as described in Remington's Pharmaceutical Science, 17th ed.,Mack Publishing Company, Easton, Pa. (1985), the entire disclosure ofwhich is herein incorporated by reference, and such pharmaceuticalcompositions may be packaged for use as a liquid, gel, cream, solid,emulsion and dispersion.

Various embodiments of the invention also include methods for using thecompositions described above. For example, some embodiments of theinvention are directed to a method for treating a biofilm including thestep of contacting the biofilm with a composition containing a surfaceactive agent and a sub-lethal amount of an antimicrobial agent, andother embodiments include a method for preventing a biofilm includingthe step of contacting biofilm forming microorganisms with a compositioncontaining a surface active agent and a sub-lethal amount of anantimicrobial agent. In some such embodiments, the biofilm may bepresent on a patient, for example, a human or a non-human animal, and inparticular embodiments, the biofilm may be part of a wound. In othersuch embodiments, the biofilm or biofilm forming microorganisms may bepresent on a biomaterial that may contact a patient. In still other suchembodiments, the biofilm or biofilm forming microorganisms may bepresent on tissue removed from a patient or tissue that is outside ofthe patient and may or may not be replaced back into the patient.

The invention also encompasses methods for using a composition includinga surface active agent and a sub-lethal amount of a antimicrobial agentfor treating surfaces. Therefore, the compositions of the invention mayhave non-pharmaceutical applications. For example, in some embodiments,a composition including a surface active agent and a sub-lethal amountof an antimicrobial agent may by applied to an inanimate object, suchas, but not limited to a chair, table, side board, machine or variousparts of a machine or combinations thereof. In other embodiments, thecompositions of the invention may be applied surgical instruments and/orsurfaces of objects in an operating room. In still other embodiments,the compositions of the invention may be applied to surfaces of devicesmeant to be implanted in a patient prior to implantation such as, amedical device, for example, a catheter.

In embodiments such as those described above, contacting the biofilm orthe biofilm forming microorganism may occur by any method known in theart. For example, in some embodiments, the composition may be applied byhand or mechanically using, for example, extrusion or spray delivery. Inthe context of embodiments that include contacting biofilms or biofilmforming microorganisms present on a patient, the composition may bedelivered by, for example, topical administration which may be performedby hand, mechanically (e.g., extrusion and spray delivery) or as acomponent in a dressing such as gauze or other wound coverings.

In embodiments in which the compositions of the invention areadministered or applied directly to a tissue or biomaterial surface byhand or mechanically, it may be important to apply the composition so asto achieve a therapeutic coating. A therapeutic coating generally refersto an amount of the composition which may form a substantially uniformcovering over the effected area and may encompass non-effected areassurrounding an injury or wound. In embodiments in which the compositionis delivered by hand, there can be considerable variation in thethickness of layers applied by practitioners. In some embodiments, atherapeutic coating may be applied or administered alone, and in otherembodiments, a therapeutic coating may be applied in combination with anoverlying dressing. In embodiments in which the composition is appliedor administered mechanically using a device that physically pushes(i.e., extrusion) or sprays the composition onto a tissue or biomaterialsurface, a uniform therapeutic coating may be achieved in a singleadministration or in several applications over the effected area, and atherapeutic coating delivered in this manner may be provided alone or incombination with an overlying dressing.

The thickness of a therapeutic coating of the composition when appliedmay vary in accordance with the size of a wound, the time available toapply the composition, the amount of composition available and othervariables. For example, in various embodiments, the thickness of theapplied compositions may be from about 1 inch thick to less than about1/10,000 inch thick or about 1 inch, about ½ inch, about ¼ inch or about1/100 inch. In some embodiments, the thickness may vary in a singleapplication. For example, the composition may be applied more thickly inthe area of a wound and less thickly in the area surrounding the wound.In other embodiments, less composition may be applied to a wound thatdoes not exhibit signs of biofilm formation while in still otherembodiments, a greater amount of the composition may be applied towounds that exhibit symptoms of biofilm formation. In yet otherembodiments, a wound and the area surrounding the wound may be coveredwith a medical fabric such as, for example, band-aids or gauze, afterbeing contacted with the composition.

The methods described above may be useful for treating patientsexhibiting a number of indications or suffering from any number ofconditions that may be susceptible to microbial biofilm formation or mayalready have a biofilm present. Such patients may be considered “in needof treatment.” In some embodiments, the indications or conditions thatmay include risk of biofilm formation and may require treatment usingthe compositions of the invention may arise from, for example, injury inwhich skin and/or skin function is disrupted or surgery. Examples ofsuch injuries may include, but are not limited to burns, abrasions,cuts, scrapes, and other denuding tissue injuries or combinations ofthese. In other embodiments, the compositions of the invention may beused to treat chronic wounds. In general, chronic wounds arecharacterized by non-healing skin wounds and include, for example,chronic venous ulcers, diabetic ulcers, arterial ulcers, pressure ulcers(e.g., decubitis ulcers), radiation ulcers, traumatic wounds, open,complicated non-healing wounds and the like.

Still other embodiments of the invention include methods for treating orpreventing a microbial biofilm including the steps of: administering afirst composition containing a surface active agent and that does notcontain an antimicrobial agent; and administering a second compositionthat contains a surface active agent and a sub-lethal amount of anantimicrobial agent. In such embodiments, the second composition maygenerally be administered after the first composition. In someembodiments, the first composition may be applied and the secondcomposition may be applied without any intervening steps, and in otherembodiments, the first composition may be applied and then removed orpartially removed by, for example, being wiped away prior toadministration of the second composition, prior to administration of thesecond composition. In still other embodiments, the method may furtherinclude the step of applying a dressing or covering to the treatedsurface following administration of either the first or second or bothcompositions. In yet another embodiment, the second composition may beincluded in the dressing material. For example, the second compositionmay be impregnated in a dressing material or otherwise contained orencompassed by the dressing material. In such embodiments, the dressingincluding the second composition may be applied over a surface to whichthe first composition has been administered. In an alternativeembodiment, the second composition may be applied directly to a dressingmaterial by, for example, coating a surface of the dressing materialwith the second composition, prior to applying the dressing to thesurface treated with the first composition.

The time between the first administration and the second administrationmay vary throughout embodiments and may be based on the patient andpractitioner. For example, in various embodiments, the time betweenadministration of the first and second compositions may be from apartial second (e.g., 0.001 seconds) up to several minutes (e.g., 1, 2,3, 4, 5, 10, 15, 20, 25 or 30 or greater than 30 minutes) or severalhours (e.g., 1, 2, 3, 4 or 5 or greater than 5 hours).

Yet other embodiments of the invention include a dressing for treatingor preventing a microbial biofilm that includes: a first layer includinga composition containing a surface active agent and no antimicrobialagent; a second layer including a second composition containing asurface active agent and a sub-lethal amount of an antimicrobial agent;and a dressing material supporting the first and second compositionlayers. In such embodiments the second layer is located between thefirst layer and the dressing material. In some embodiments, the secondlayer may form a coating covering a surface of the dressing material,and in other embodiments, the second layer is impregnated or otherwisecontained within or encompassed by the dressing material. In certainembodiments, the first and second composition layers may completelyencompass the dressing, and in others, the first and/or secondcomposition layers may be placed such that the compositions may bepositioned to be delivered to only a portion of the patient to which thedressing is applied. For example, in one embodiment, the entire dressingincludes the first and second composition layers, and in anotherembodiment, the dressing may include a middle section that includesfirst and second composition layers that is flanked on either side byportions of the dressing that do not contain composition layers. Instill another embodiment, the entire dressing may contain a firstcomposition layer and a portion of the dressing may contain the secondcomposition layer.

In still other embodiments, the dressing may further include a spacerlayer between the first and second composition layers. In someembodiments, the spacer layer may be a dressing material that allows theantimicrobial agent to be brought into contact with the patient afterthe dressing has been applied to the patient. In other embodiments, thespacer layer may be a pharmaceutical agent such as, for example, apolymer, a cream, a wax and the like that may separate the first andsecond compositions. In certain embodiments, the spacer layer may loseits integrity by, for example, disintegrating, dissipating, becomingporous, etc., upon application of the dressing to a patient. Forexample, in one embodiment, the spacer layer may degrade as it is warmedto body temperature. In general as the spacer layer loses integritymeans, it may no longer function as a barrier between the first andsecond composition. Thus, the second composition may come into contactwith patient and/or the first composition as the spacer layer losesintegrity.

The dressing material of embodiment may be any pharmaceuticallyacceptable fabric. For example, in various embodiments, the dressingmaterial may be gauze, a gauze pad, polymeric or natural fiber band-aid,second skin or any other type of material or fabric useful in themedical arts to cover a wound or at least keep a therapeutic agent orpharmaceutical composition in contact with a patient.

The compositions of the invention may be packaged in any way whichallows a practitioner or an injured individual access to the compositionfollowing injury. For example, in one embodiment, the first and/orsecond compositions are contained within a tube or bottle from which thecomposition may be poured and applied to the injury, and in anotherembodiment, the first and/or second composition may be absorbed onto aswab which may be used to apply the compositions of the invention. Instill another embodiment, the first and/or second composition may becontained within a vial that is broken to release the composition whichmay then be applied by means discussed herein above. Of course, otherpackaging means are available and may be used in conjunction withembodiments of the invention.

Various embodiments of the invention described above may prevent biofilmformation. In such embodiments, the patient or wound may not exhibitsigns or symptoms of biofilm infestation. However, microorganisms withthe potential to form a biofilm may be present on the patient or withinthe wound itself. The compositions of the invention may eliminate thebiofilm forming microorganisms before biofilm formation has if thecomposition is applied to a wound prior to formation of a biofilm. Asdescribed above, biofilm forming microorganisms begin to form a biofilmonly when a population of planktonic microorganisms reach a specificcell density and/or when the concentration of microorganism producedautoinducer has reached a threshold level. The concentration ofautoinducer and cell density requirements may vary among biofilm formingspecies. Without wishing to be bound by theory, application of thecompositions of the invention prior to formation of a biofilm may reducethe microorganism population such that planktonic microorganisms may notreach an adequate cell density to form a biofilm thereby inhibitingbiofilm formation.

Because biofilms may form rapidly, it may be advantageous for emergencypersonnel (i.e., first responders) to apply a composition according tothe present invention at the scene of the injury. Thus, in anotherembodiment, the present invention provides a method for treating awounded patient by administering to the wounded patient a compositioncontaining a surface active agent and a sub-lethal amount of anantimicrobial agent within about 10 hours of injury. In otherembodiments, the wound may be treated within less than 10 hours. Forexample, a wound may be treated within 8 hours, 5 hours, 4, hours, 3hours, 2 hours or 1 hour or within 30 minutes, within 10 minutes orwithin 5 minutes. Without wishing to be bound by theory, treating aninjury within about 2 hours or less may ensure that the wound is treatedwhile the biofilm forming pathogens in the wound are in a planktonicstate or before biofilm forming pathogens have invaded the wound. Thus,any biofilm forming pathogens are contacted by the composition of theinvention before the concentration of autoinducer has reached athreshold level to induce biofilm formation and/or before the celldensity of the pathogen has reached a similar threshold level.Accordingly, formation of a biofilm may be reduced or eliminated, andthe wound may be more easily treated.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Thisinvention encompasses all combinations of the different aspects of theinvention noted herein. It is understood that any and all embodiments ofthe present invention may be taken in conjunction with any otherembodiment or embodiments to describe additional more preferredembodiments. It is also to be understood that each individual element ofthe preferred embodiments is intended to be taken individually as itsown independent preferred embodiment. Furthermore, any element of anembodiment is meant to be combined with any and all other elements fromany embodiment to describe an additional embodiment.

This invention and embodiments illustrating the method and materialsused may be further understood by reference to the followingnon-limiting examples.

EXAMPLES

Overview

Compositions prepared in accordance with embodiments of the inventionwere evaluated for efficacy using “The Calgary Biofilm Device” asdescribed in Ceri et al. “The Calgary Device: New Technology for theRapid Determination of Antibiotic Susceptibilities of BacterialBiofilms.” J. Clin. Microbio. 1999; 37:1771-1776 hereby incorporated byreference in its entirety. Briefly, this simple assay allowsmicroorganisms to grow on pins which are positioned on the lid of a 96well microtiter plate and which protrude into the wells of the 96 wellmicrotiter plate when the lid is positioned on the plate. Biofilmforming microorganisms are placed on the pins and contact growth mediaand/or test compounds in the wells of a microtiter plate. This allowsrapid analysis of test compounds efficacy against biofilm formingmicroorganisms. The data resulting from the assay produces both MIC andMBEC values by observing biofilm growing on the pegs. Similar assays mayalso be used to determine the MIC and MBEC of planktonic microorganismsby growing the microorganisms suspended in the wells of a 96 wellmicrotiter plate, and observing the growth of the microorganisms inwells that contain the test compound and comparing these values withcontrol well that do not contain a test compound.

The assay described above compounds according to various embodiments ofthe invention were tested against biofilm forming microorganisms:Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213),and Staphylococcus epidermidis (ATCC 35984). These microorganisms weregrown in media containing various compositions of the inventionincluding:

Poloxamer 188 dissolved in water to concentrations of 45%, 50% and 55%by volume poloxamer 188.

PSSD (50% poloxamer 188 and 1% silver sulfadiazine in water) at 100% anddiluted with 50% poloxamer 188 in water to concentrations of 90%, 80%,70%, 60% and 50% by volume PSSD.

PTAG (50% poloxamer 188 and TAG (Polymyxin B [10,000 U/g],nitrofurantoin [0.3%], nystatin [4,000 U/g]) in water) at 100% anddiluted with 50% poloxamer 188 in water to concentrations of 90%, 80%,70%, 60% and 50% by volume PTAG.

SSD cream (1% silver sulfadiazine) at 100% and diluted in cream toconcentrations of 90%, 80%, 70%, 60% and 50% by volume SSD.

The results provided below show that all of compounds of the inventiontested had some degree of antimicrobial biofilm activity and varioustested compounds of the invention were able to both prevent biofilmformation and eradicate preformed biofilms. Generally, this activity isimproved over the activity of silver sulfadiazine observed inside-by-side comparisons.

Inhibition of Preformed Biofilms

An inoculum of the microorganism to be tested was prepared to match a0.5 McFarland Standard (1.5×10⁸ cells per ml) in 3 ml of sterile waterin a glass test tube using a sterile cotton swab. This inoculum was thendiluted 1 in 30 in Cation Adjusted Mueller Hinton Broth (CAMHB) andinverted 3-5 times to achieve uniform mixing of the microorganism. Asample (100 μl) of the diluted inoculum was serially diluting and spotplating on TSA to confirm the cell density of the sample. 150 μl of theremaining diluted inoculum was then placed in each well of a 96 wellNUNC bottom microtiter plate, and a lid including 96 pegs was placed onthe plate. The device (i.e., the microtiter plate and corresponding pegcontaining lid) was placed on a shaker in a humidified incubator at 37°C. for 24 hours set at 110 revolutions per minute to allow biofilm onthe peg.

Following incubation, the lid was removed from the device and the pegswere rinsed in sterile saline to remove planktonic cells from thebiofilm that formed on the plate. The peg containing lid was thentransferred to challenge plate, and the challenge plate was incubated at37° C. for a specified contact time. A contact plate is a sterile96-well microtiter plate whose wells contain a concentration of the testcomposition as specified above.

Surviving biofilm forming microorganisms were recovered by rinsing thepegs in 0.9% sterile saline and then transferring the pegs to a recoverymedia which was then sonicated to dislodge surviving biofilm formingbacteria from the peg. The recovery plate was then incubated for 24hours at 37° C., and the cell density was determined by obtaining anoptical density at 630 nm (OD₆₃₀). In the tables below, clear wellshaving an OD₆₃₀ less than 0.1 are evidence that the biofilm formingbacteria were eradicated and denoted by a minus (−). Wells in whichbacterial growth is evident (OD₆₃₀ greater than 0.1) are denoted with aplus (+).

Inhibition of Biofilm Formation

A inoculum as described above were prepared, diluted 1 in 30 in CationAdjusted Mueller Hinton Broth (CAMHB), inverted 3-5 times to achieveuniform mixing of the microorganism and sample (100 μl) of the dilutedinoculum was serially diluting and spot plating on TSA to confirm thecell density of the sample. A challenge plate was prepared as describedabove, and 20 μl of the inoculum was added to each well of the challengeplate. A sterile lid having 96 pegs corresponding to each of the wellsof the challenge plate was placed over the challenge plate and thedevice was incubated in a humidified incubator at 37° C. for 24 hoursshaking at 110 revolutions per minute to allow biofilm on the peg for aspecified contact time. The plate was then removed from incubation, andsurviving biofilm forming bacteria on each peg were recovered asdescribed above. Following incubation, the cell density was determinedby obtaining an optical density at 630 nm (OD₆₃₀). In the tables below,clear wells having an OD₆₃₀ less than 0.1 are evidence that the biofilmforming bacteria were eradicated and denoted by a minus (−). Wells inwhich bacterial growth is evident (OD₆₃₀ greater than 0.1) are denotedwith a plus (+).

MBC and MBEC

The minimum bactericidal concentration (MBC) value represents the lowestconcentration which kills 99.9% of the population. Results weredetermined following the 24 hour incubation from the Test panels usingthe plate reader. To determine MBC, the optical density of the wells ofeach challenge plate was determined at 630 nm (OD₆₃₀). Clear wells(OD₆₃₀<0.1) are evidence of inhibition and denoted (−). Wells in whichbacterial growth is evident (OD₆₃₀ greater than 0.1) are denoted with aplus (+).

The minimum biofilm eradication concentration (MBEC) was determinedfollowing the 24 hour incubation from the MBEC panels using the platereader in conjunction with Log10 reduction data. To determine MBEC, theoptical density of the wells of the recovery plate was determined at 630nm (OD₆₃₀). Clear wells (OD₆₃₀<0.1) are evidence of biofilm eradicationand denoted (−). The MBEC is defined as the minimum concentration ofantibiotic that inhibits growth of the biofilm. Wells in which bacterialgrowth is evident (OD₆₃₀ greater than 0.1) are denoted with a plus (+).

Example 1 P. aeruginosa

An inoculum of P. aeruginosa was prepared as described above and testedin an inhibition of biofilm assay to yield the following results:

MBC  55% 50% 45% P. 188 − − + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD − − − − − − MBEC  55% 50% 45% P. 188 − − + 100% 90%80% 70% 60% 50% PSSD + − + + − − TAG − − + − + − SSD + + − + − +

An inoculum of P. aeroginosa was prepared as described above and testedin an inhibition of preformed biofilm assay to yield the followingresults:

MBC  55% 50% 45% P. 188 − − + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD − − − + − − MBEC  55% 50% 45% P. 188 − + + 100% 90%80% 70% 60% 50% PSSD + − + + + − TAG + + + + + + SSD + + + + + +

Example 2 S. aureus

An inoculum of S. aureus was prepared as described above and tested in ainhibition of biofilm assay to yield the following results:

MBC  55% 50% 45% P. 188 − + + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD + + + + − − MBEC  55% 50% 45% P. 188 + + + 100% 90%80% 70% 60% 50% PSSD − − + + + − TAG + − + − − − SSD + + + + + +

An inoculum of S. aureus was prepared as described above and tested inan inhibition of preformed biofilm assay to yield the following results:

MBC  55% 50% 45% P. 188 − + + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD + + + + − − MBEC  55% 50% 45% P. 188 + + + 100% 90%80% 70% 60% 50% PSSD + − + + + − TAG + − + + + + SSD + + + − − −

Example 3 S. epidermidis

An inoculum of S. epidermidis was prepared as described above and testedin a inhibition of biofilm assay to yield the following results:

MBC  55% 50% 45% P. 188 + + + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD + + + + + + MBEC  55% 50% 45% P. 188 − + + 100% 90%80% 70% 60% 50% PSSD − − + + + − TAG + − − − − − SSD + + + + + +

An inoculum of S. epidermidis was prepared as described above and testedin an inhibition of preformed biofilm assay to yield the followingresults:

MBC  55% 50% 45% P. 188 + + + 100% 90% 80% 70% 60% 50% PSSD − − − − − −TAG − − − − − − SSD + + + + + + MBEC  55% 50% 45% P. 188 − − + 100% 90%80% 70% 60% 50% PSSD + − + + + + TAG + − + − + − SSD + + + + + +

What is claimed is:
 1. A method for treating a microbial biofilm on a patient comprising contacting the microbial biofilm with a composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent.
 2. The method of claim 1, wherein the antimicrobial agent is selected from an antibacterial, antifungal, antiviral or combinations thereof.
 3. The method of claim 1, wherein the antimicrobial agent is selected from furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin, silver sulfadiazine, nanocrystalline silver, ionic silver, honey, iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine and combinations thereof.
 4. The method of claim 1, wherein a sub-lethal amount of an antimicrobial agent is a standard therapeutically effective amount.
 5. The method of claim 1, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 1% by weight of the composition.
 6. The method of claim 1, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.95% by weight of the composition.
 7. The method of claim 1, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.75% by weight of the composition.
 8. The method of claim 1, wherein the surface active agent is selected from a poloxamer, meroxapol, poloxamine and combinations thereof.
 9. The method of claim 1, wherein the surface active agent is selected from a poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer 407 and combinations thereof.
 10. The method of claim 1, wherein the surface active agent is poloxamer
 188. 11. The method of claim 1, wherein contacting the microbial biofilm comprises applying the composition to a wound.
 12. The method of claim 1, wherein contacting the microbial biofilm comprises administering the composition topically.
 13. The method of claim 12, wherein administering the composition topically is selected from administering by hand, administering by an extruder, spray delivery, applying a dressing including the composition and combinations thereof.
 14. The method of claim 1, wherein contacting the microbial biofilm comprises contacting tissue from the patient that is outside the patient.
 15. The method of claim 1, wherein contacting comprises applying the composition to a dressing prior to applying the dressing to the patient.
 16. The method of claim 1, wherein the patient is afflicted with an injury.
 17. The method of claim 16, wherein the injury is selected from a burn, abrasion, cut, scrape, denuding tissue injury and combinations thereof.
 18. The method of claim 1, wherein the patient is afflicted with a chronic wound.
 19. The method of claim 18, wherein chronic wound is selected from a venous ulcer, diabetic ulcer, arterial ulcer, pressure ulcer, radiation ulcer, traumatic wound, non-healing wound and combinations thereof.
 20. A method for preventing a microbial biofilm on a patient comprising administering a composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent to an injury.
 21. The method of claim 20, wherein the antimicrobial agent is selected from an antibacterial, antifungal, antiviral or combinations thereof.
 22. The method of claim 20, wherein the antimicrobial agent is selected from furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin, silver sulfadiazine, silver salts, iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine and combinations thereof.
 23. The method of claim 20, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 1% by weight of the composition.
 24. The method of claim 20, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.95% by weight of the composition.
 25. The method of claim 20, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.75% by weight of the composition.
 26. The method of claim 20, wherein the surface active agent is selected from a poloxamer, meroxapol, poloxamine and combinations thereof.
 27. The method of claim 20, wherein the surface active agent is a copolymer selected from a poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer 407 and combinations thereof.
 28. The method of claim 20, wherein the surface active agent is poloxamer
 188. 29. The method of claim 20, wherein administering comprises applying the composition to a wound.
 30. The method of claim 20, wherein administering comprises administering the composition topically.
 31. The method of claim 30, wherein administering the composition topically is selected from administering by hand, administering by an extruder, spray delivery, applying a dressing including the composition and combinations thereof.
 32. The method of claim 20, wherein administering comprises contacting tissue from the patient that is outside the patient.
 33. The method of claim 20, wherein administering comprises applying the composition to a dressing prior to applying the dressing to the patient.
 34. The method of claim 20, wherein the injury is selected from a burn, abrasion, cut, scrape, denuding tissue injury and combinations thereof.
 35. The method of claim 20, wherein the composition is administered while biofilm forming pathogens are in a planktonic state.
 36. A method for treating or preventing a microbial biofilm in a patient comprising: administering a first composition comprising a surface active agent to an injury wherein the first composition does not contain an antimicrobial agent; and administering a second composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent to the injury; wherein the first composition is administered before the second composition.
 37. The method of claim 36, wherein the second composition is applied to a dressing prior to its administration.
 38. The method of claim 36, wherein the antimicrobial agent of the second composition is selected from an antibacterial, an antifungal, an antiviral or a combination thereof.
 39. The method of claim 36, wherein the antimicrobial agent of the second composition is selected from furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin, silver sulfadiazine silver salts, nanocrystalline silver, ionic silver, iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine and combinations thereof.
 40. The method of claim 36, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 1% by weight of the composition.
 41. The method of claim 36, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.95% by weight of the composition.
 42. The method of claim 36, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.75% by weight of the composition.
 43. The method of claim 36, wherein the surface active agents in the first and second composition are surfactants.
 44. The method of claim 36, wherein the surface active agents in the first and second compositions are independently selected from poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer 407 and combinations thereof.
 45. The method of claim 36, wherein the surface active agents in the first and second compositions are the same.
 46. The method of claim 36, wherein the surface active agents in the first and second compositions are poloxamer
 188. 47. A composition for treating or preventing a microbial biofilm comprising: a surface active agent; and a sub-lethal amount of an antimicrobial agent.
 48. The composition of claim 47, wherein the antimicrobial agent is selected from an antibacterial, an antifungal, an antiviral or a combination thereof.
 49. The composition of claim 47, wherein the antimicrobial agent is selected from furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin, silver sulfadiazine, nanocrystalline silver, ionic silver, silver salts, iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine, honey and combinations thereof.
 50. The composition of claim 47, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 1% by weight of the composition.
 51. The composition of claim 47, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.95% by weight of the composition.
 52. The composition of claim 47, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.75% by weight of the composition.
 53. The composition of claim 47, wherein the composition further comprises a solvent.
 54. The composition of claim 47, wherein the surface active agent is selected from a poloxamer, meroxapol, poloxamine and combinations thereof.
 55. The composition of claim 54, wherein the poloxamer is selected from a poloxamer 101, poloxamer 105, poloxamer 105 benzoate, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 182 dibenzoate, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403 and poloxamer 407 and combinations thereof. Agent is a copolymer selected from poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer 407 and combinations thereof.
 56. The composition of claim 53, wherein the surface active agent is poloxamer
 188. 57. The composition of claim 54, wherein the meroxapol is selected from a meroxapol 105, meroxapol 108, meroxapol 171, meroxapol 172, meroxapol 174, meroxapol 178, meroxapol 251, meroxapol 252, meroxapol 254, meroxapol 258, meroxapol 311, meroxapol 312, meroxapol 314 and combinations thereof.
 58. The composition of claim 54, wherein the poloxamine is selected from a poloxamine 304, poloxamine 504, poloxamine 701, poloxamine 702, poloxamine 704, poloxamine 707, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1101, poloxamine 1102, poloxamine 1104, poloxamine 1301, poloxamine 1302, poloxamine 1304, poloxamine 1307, poloxamine 1501, poloxamine 1502, poloxamine 1504, poloxamine 1508 and combinations thereof.
 59. The composition of claim 47, further comprising one or more secondary active agents.
 60. The composition of claim 47, further comprising one or more additives.
 61. A dressing for treating or preventing a microbial biofilm comprising: a first composition layer comprising a surface active agent wherein the first gel layer does not include an antimicrobial agent; a second composition layer comprising a surface active agent and a sub-lethal amount of an antimicrobial agent; and a dressing material supporting said first and second composition layers; wherein the second composition layer is located between the first composition layer and the dressing material.
 62. The dressing of claim 61, wherein the antimicrobial agent is selected from an antibacterial, an antifungal, an antiviral or a combination thereof.
 63. The dressing of claim 61, wherein the antimicrobial agent is selected from furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin, furazolidone, 2-(methoxymethyl)-5-nitrofuran, nidroxyzone, nifuroxime, nifurazide, nitrofurazone, nystatin, polymyxin, silver sulfadiazine, nanocrystalline silver, ionic silver, silver salts, iodine, benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine, honey and combinations thereof.
 64. The dressing of claim 61, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 1% by weight of the composition.
 65. The dressing of claim 61, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.95% by weight of the composition.
 66. The dressing of claim 61, wherein the antimicrobial agent is silver sulfadiazine and the sub-lethal amount is less than about 0.75% by weight of the composition.
 67. The dressing of claim 61, wherein the surface active agent in the first and second compositions is selected from a poloxamer, meroxapol, poloxamine and combinations thereof.
 68. The dressing of claim 61, wherein the surface active agent in the first and second compositions is independently selected from a poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 335, poloxamer 407 and combinations thereof.
 69. The dressing of claim 61, wherein the surface active agent in the first and second compositions is poloxamer
 188. 70. The dressing of claim 61, wherein the surface active agent in the first and second compositions is the same.
 71. The dressing of claim 61, further comprising a spacer material layer between said first and second composition layers.
 72. The dressing of claim 71, wherein the spacer material fully or partially loses integrity upon application of the dressing to a patient.
 73. The dressing of claim 61, wherein the second composition layer impregnates the dressing material.
 74. A method for treating or preventing a microbial biofilm on a patient comprising administering a composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent to a wound prior to infection.
 75. A method for treating or preventing a microbial biofilm on a patient comprising administering a composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent to a wound while biofilm forming bacteria are in a planktonic state.
 76. A method for treating or preventing a microbial biofilm on a patient comprising administering a composition comprising a surface active agent and a sub-lethal amount of an antimicrobial agent to a wound prior to infection wherein the composition is administered within 10 hours of the injury.
 77. A method for treating or preventing a microbial biofilm comprising administering a composition comprising a surface active agent to a wound. 